FIGURE 1.

TPC2 is a functional ion channel activated by NAADP. a, anti-HA immunoblot of protein incorporated into bilayers. Sol corresponds to solubilized sample before IP, Specific corresponds to IP with anti-HsTPC2 serum, and Nonspecific corresponds to a control IP with non-immune rabbit serum. b and c, a typical single-channel experiment showing sequential additions of NAADP to the trans and cis chambers with K⁺ (b) or Ca²⁺ (c) as permeant ion. d and e, current-voltage relationships with K⁺ or Ca²⁺, respectively, as permeant ion. The dotted line in d illustrates the KCl gradient (trans, 210 mm; cis, 510 mm) data. f, the relative permeability of TPC2 to K⁺ and Ca²⁺. The single-channel current-voltage relationship with 210 mm KCl in the cis chamber and 210 mm CaCl₂ in the trans chamber yields a reversal potential of 26 ± 1 mV (n = 3). The PCa²⁺/PK⁺ was calculated to be 2.6 ± 0.17. The bi-ionic conductance over the range −30 to +60 mV (22 ± 3 pS (n = 3)) is similar to that obtained using only Ca²⁺ as the permeant ion (15 ± 1.5 pS) as expected if Ca²⁺ is the ion predominantly flowing through the channel at potentials close to 0 mV.